A major obstacle to HIV-1 eradication is the existence of latently infected cells that persist despite antiretroviral therapy (ART). Current eradication strategies focus on the reactivation and clearance of infected cells with various latency reactivating agents (LRA). However, viral reactivation alone is insufficient to reduce HIV-1 DNA reservoirs, and the association between increased cell-associated HIV-1 RNA and the frequency of reactivated cells is poorly understood. It is also unknown how reactivation strategies and HIV-1 genome integrity affect the frequency or amplitude of HIV-1 transcription. As a result, we have developed and implemented a novel method that provides insights into HIV-1 persistence that are inaccessible though existing technologies. Individual cells are encapsulated into nanoliter-scale reaction droplets, followed by intra-droplet lysis and PCR amplification of unspliced (us) and multiply spliced (ms) HIV-1 RNA and downstream isolation and sequencing of genomic viral DNA. We have successfully applied this method to identify transcriptionally active CD4+ T cells from HIV-1-infected patients on suppressive ART with and without LRA stimulation. Our data suggest the frequency of infected cells may increase while total cell-associated levels decrease and vice versa. We also observed dichotomies between usRNA and msRNA responses to latent reservoir activation. These findings suggest that single-cell analysis will be crucial in providing insight into which cells and tissues are targets for eradication in shock and kill approaches. We propose to utilize our assay to perform high-throughput reservoir quantification and downstream HIV-1 genome characterization of cells isolated from latently-infected peripheral blood and organized lymphoid tissue obtained from early and late ART treated individuals. We hypothesize that effector cells will display higher frequencies and amplitudes of HIV-1 RNA reactivation than those with memory or regulatory phenotype. Early treated individuals are thought to have smaller reservoirs, observed preferentially in effector memory cells which may have the propensity to reactive HIV-1 more efficiently. Our aims are to: (1) determine HIV-1 RNA transcriptional activity in response to various HIV-1 latency reversing agents in single cells derived from peripheral blood and organized lymphoid tissues from suppressed patients, (2) determine the single-cell responses to ex vivo LRA reactivation in early and late antiretroviral treated individuals, and (3) define the relationship between single-cell HIV-1 genetic sequence integrity, and HIV-1 transcriptional activity. In future studies, we plan to apply our novel methods to characterize the in vivo responses to interventional trials of HDACi and other immune modulating therapies. We also plan to adapt our assay to directly measure viral outgrowth in individual cells.